Elevated circulatory cholesterol is an established risk factor in the development of atherosclerosis. Atherosclerosis can be described as the process of the weakening of arterial walls and a narrowing of the blood flow within these vessels. This sequence of events frequently occurs in the coronary arteries, causing blockage of blood flow to the heart and leading to myocardial infarction (heart damage); it is often termed coronary heart disease (CHD).
Therapeutic agents, such as statins, which control the concentration of serum cholesterol, have shown some effectiveness in the treatment of coronary heart disease. These agents modulate circulating levels of cholesterol-carrying, lipoproteins by inhibiting cholesterol synthesis itself, but have no effect on downstream events such as cholesterol absorption or oxidation, which are necessary steps for initiating atherosclerosis. However, cholesterol levels are constantly under upward pressure due to high dietary intake of animal fat, and on account of the body's synthesis of cholesterol in the liver and other tissue when dietary supply is deemed inadequate.
Four major classes of lipoproteins are known, all of which share a similar basic structure of a lipid nucleus surrounded by an amphiphilic surface layer of phospholipids and apolipoproteins. The larger the lipid nucleus the less dense is the lipoprotein particle. In increasing order of size they are: high density lipoprotein (HDL), which develops a lipid core by scavenging cholesterol from peripheral tissue and hence is often referred to as “good cholesterol”; low density lipoprotein (LDL), which forms in the liver from very low density lipoprotein (VLDL) remnants; very low density lipoproteins (VLDL), which are also made in the liver and contain mostly triglycerides and chylomicrons which are formed in the small intestine and are made up of triglycerides and dietary fat.
A number of patents disclose compounds which are useful anti-atherosclerotic agents. For example, U.S. Pat. No. 4,681,893, which discloses a “Method of Inhibiting cholesterol biosynthesis in a patient,” and U.S. Pat. No. 5,846,966, which discloses “Combinations of hydroxy-substituted azetidinone compounds and HMG CoA Reductase Inhibitors,” This patent (U.S. Pat. No. 4,681,893) and all other patents and patent applications mentioned in the present application are hereby incorporated herein by reference.
More recent evidence supports the belief that LDL needs to undergo a series of transformations and complexation prior to the onset of atherosclerosis. A putative early step is the formation of oxidized LDL (oxLDL), which plays a pivotal role in the onset of atherosclerosis. Oxidative modification of LDL alters its biological properties, resulting in chemotaxis of monocytes or T lymphocytes in addition to the modulation of growth factors and cytokine production from endothelial cells, smooth muscle cells, and macrophages. The cytotoxicity oxLDL from cultured endothelial cells has been clearly demonstrated to be atherogenic. These studies clearly showed the atherogenic mechanisms of oxidation of LDL in arterial walls but later studies have begun to put more emphasis on the clinical importance of circulating oxLDL levels in patients with coronary heart disease. This circulating oxLDL is measured with an immunoassay using murine monoclonal antibodies prepared against malondialdehyde-modified LDL (MDA-LDL) and against copper oxLDL.
More recent trials have also shown the presence of increased plasma levels oxLDL in patients with coronary heart disease. Elevated plasma oxLDL levels have been established as a biochemical risk marker for CHD. The absence of association of oxLDL levels with other risk factors, such as hypertension, hyperlipidemia, or smoking suggests that raised oxLDL levels are an independent risk factor for CHD and when compared with other biochemical markers, such as total cholesterol, triglycerides, apoB, or HDL levels, the association between oxLDL, levels and CHD is a markedly superior risk indicator for CHD.
The uptake of oxLDL by endothelial cells has also been shown to be a critical step for the initiation and development of atherosclerosis. Adhesion molecules are inflammatory markers, which are up-regulated by oxLDL and play a pivotal role in atherogenesis. Another manner in which oxLDL may be shown to contribute to CHD is by assisting in the accumulation of apoptotic cells in atherosclerotic plaques.
Fish oil and more specifically the Eicosa Pentaenoic Acid (EPA), Docosa Pentaenoic Acid (DPA), and Docosa Hexaenoic Acid (DHA) polyunsaturated fatty acid (PUFA) constituents of fish oil have been shown to have potential use as a cardiotonic stimulant. Some in-vitro studies carried out in human coronary endothelial cells have suggested that EPA and DHA may attenuate expression of adhesion molecules which may be one pathway for an anti-atherosclerotic effect of fish and fish oils.
In another instance, the influence of dietary fish oil on aortic thrombosis, platelet aggregation, and superoxide dismutase (SOD) activity in a rat model has been studied and shown to delay formation of arterial thrombus, probably by reducing platelet aggregation and oxidative stress-associated arterial injury.
It is also well established in mono-phasic chemical systems that the highly unsaturated EPA and DHA should oxidize more readily than fatty acids that contain fewer double bonds. Previous studies have showed that enrichment of LDL, which has discrete polar and non-polar phases, with these fatty acids did not increase oxidation.
Studies by Wander (Lipids, Vol. 37, No. 8, pg 789, (2002)) have also shown that the extent of apoptosis induced by EPA/DHA-rich oxLDL compared to that induced by EPA/DHA-non-rich oxLDL in U937 cells is significantly lower thus leading to the conclusion that after PUFA supplementation, EPA/DHA-rich oxLDL-induced cell apoptosis decreased and the decrease was not related to the concentration of lipid hydroperoxides. This result suggests that EPA/DHA provides for a protective effect for atherosclerosis via a lessening of cell apoptosis in the arterial wall, rather than any reduction in oxidative damage.
Atherosclerosis has also been characterized by a gradual thickening of arterial walls due to the excessive accumulation of lipids. Pro-inflammatory factors and dyslipidemia are the main contributors to its development as described by Steinberg D., J. Biol. Chem. Vol. 272(34), pg. 20963, (1997) and Steinberg D., Nature Med. Vol. 8, pg. 1211, (2002). Low density lipoprotein (LDL) is the principal form of cholesterol that accumulates in atherosclerotic lesions or plaques, but LDL must be first modified into an oxidized structure (oxLDL) to begin the process, and as shown by McMurray H. et al. J. Clinical. Med. Vol. 92, pg. 1004, (1993) the most significant pro-atherogenic mechanism for modifying LDL into oxLDL is oxidative stress.
Unlike native LDL, oxLDL binds to β2GPI-glycoprotein to eventually form a stable non-dissociable complex. (oxLDL-β2GPI complex, oxidized low density lipoprotein-beta-2-glycoprotein 1 complex) These stable complexes are regarded as pathogenic and highly clinically relevant and have been implicated as pro-atherogenic antigens and represent a serologic risk factor to the development of athero-thrombosis.
The Role of MPO
Myeloperoxidase (MPO) is a heme peroxidase-cyclooxygenase enzyme expressed in neutrophils, monocytes and macrophages. MPO participates in the innate immune defence system by forming microbicidal reactive oxidants such as hypochlorous acid, a potent antimicrobial agent. However, hypochlorous acid has also been reported to react with nucleobases resulting in the formation of 5-chlorouracil, a marker for DNA damage during inflammation, which is enriched in human atherosclerotic tissue.
Recently evidence has emerged that MPO-derived oxidants contribute to tissue damage and the initiation and propagation of acute and chronic vascular inflammatory disease. Circulating levels of MPO have been shown to predict risks for major adverse cardiac events and specific levels of MPO-derived chlorinated compounds are known biomarkers for atherosclerosis disease progression. (Takeshita J, Byun Nhan T Q, Pritchard D K, Pennathur S, Schwartz S M et al. (2006). Myeloperoxidase generates 5-chlorouracil in human atherosclerotic tissue: a potential pathway for somatic mutagenesis by macrophages, J. Biol. Chem., 281: 3096-3104)
Another indicator for MPO-catalysed oxidation of lipid proteins is observed by hydrochlorous acid attack and formation of 3-chlorotyrosine which has been identified in human atherosclerotic lesions and lipoproteins extracted from these lesions (Hazen S L, Heinecke J W, 3-Chlorotyrosine, a specific marker of myeloperoxidase-catalyzed oxidation, is markedly elevated in low density lipoprotein isolated from human atherosclerotic intima. J Clin Invest 99: 2075-2081 (1997); Thuktani. A K, McHowat J, Hsu F F, Brennan M L, Hazen S L, Ford D A, Identification of alpha-chloro fatty aldehydes and unsaturated lysophosphatidylcholine molecular species in human atherosclerotic lesions. Circulation 108: 3128-3133 (2003)).
Surprisingly, the present inventors have found that patients using a cooking oil formulation, containing fish and/or algal oil rich in mixed PUFA's, in their daily diet, over a sustained period, showed a significant reduction in circulatory oxLDL-β2GPI complex. The positive effect of the cooking oil formulation cannot be derived from the known and described literature.
Further surprisingly, the present inventors followed up this unexpected result with a second trial using a once-a-day dose of fish oil capsule containing a mixture of PUFA's and showed the same surprising lowering of circulatory oxLDL-β2GPI complex levels as observed in the cooking oil trial. In a further extension of this result, the present inventors carried out a comparative trial between an oil containing predominantly a single PUFA (DHA) and an oil containing a mixture of PUFA's (DHA, EPA, DPA) and showed the surprising statistically significant additional lowering of OxLDL-β2GPI complex levels for the mixed PUFA's as compared to oil containing predominantly a single PUFA.
In a fourth trial, the present inventors discovered a surprising, statistically significant, lowering of circulatory MPO levels for patients taking an oral dose of mixed PUFA's as compared to an untreated control group.